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Gene Therapy for Leber's Congenital Amaurosis
LCA Overview Leber's Congenital Amaurosis (LCA) is a hereditary retinal degeneration condition that has been successfully cured by gene therapy in 2008. It is an autosomal recessive trait that is known to have mutations in up to 15 different genes that are all needed for normal vision. The genes that can be affected are genes that are responsible for photoreceptors, phototransduction, and even cilia that play a role in sensory input for the eye. The four most common genes associated with LCA are CEP290, CRB1, GUCY2D, and RPE65. The most prevalent mutated gene that accounted for 15% to 22% of LCA was CEP290 (Centrosomal Protein 290-kD), which is a gene that codes for a ciliary assembly protein. Clinical symptoms are seen very early, typically around seven months of age when there is significant rod and cone cell degeneration. This will result in complete blindness by the time the person reaches five years old. LCA due to a CEP290 mutation is also known as LCA10. The Genetic Deficiency of CEP290 The genetic deficiency is a mutated CEP290 gene with a splicing defect. An exon containing a premature stop codon is inserted into the mRNA because of a mutation (c.2991+1655A→G) at chromosome location 12q21.3 of intron 26. The CEP290 gene is responsible for ciliogenesis for the retina so when a mutation in this gene happens, the protein created is shortened and the cilia do not function. This is what causes the retinal dystrophy. The CEP290 gene effects the connecting cilium, which is the transition zone of photoreceptors that connects the inner and outer layers of rod and cone cells. Therefore, this results in a failure of signal transmission from light stimuli to chemical signals transmitted to the brain. The blindness is due to this specific disconnect in the visual pathway, from the time light enters the eye to the time the signals reach the brain and undergo cognition. Gene Therapy Strategy The utilization of mice in genetics and genomics has led to many successful experiments because of the homology between the mouse genome and the human genome. Using transgenic CEP290, it was found that mouse CEP290 did not compromise the expression of the protein in the human eye in some cases. The splicing site recognition in the mouse and in the human are different, which led to the understanding that mice may not be the best organism to generate consistent results of this CEP290 mutation. Despite this, the RPE65 gene was successfully "fixed" by using an adeno-associated virus (AAV) to package the gene and inject the DNA into the host cell's genome. This encouraged the usage of AAV for CEP290, however, the CPE290 gene is too big to fit into AAV. In order to insert the 8kb CEP290 gene into a human host cell through viral transmission, the Lentivirus is used because it is large enough to hold the gene. The Cytomegalovirus (CMV) promoter was used to create efficient CEP290 packaging. The packaged "unit" is termed LV-CMV-hCEP290, and is transduced to specific cells types for gene expression. Using iPSC's (induced Pluripotent Stem Cells) and patient specific fibroblasts to obtain photoreceptor precursor cells, the functional gene can be inserted into the genome and will be expressed. Once this happens the light signals are transmitted to the brain effectively and are proven to reactivate the brain centers used for interpreting vision Associated Consequences Although somatic cell gene therapy has proven to be a functional method for treating LCA, there are a couple factors that must be taken into account. It is known that over-expression of the CEP290 gene can be cytotoxic, which is clearly an undesired reaction to treatment. The amount of gene expression by CEP290 must be known and understood very well to create positive results.With that said, the effectiveness of this gene therapy method is dependant on the patients condition at the time of treatment. It is essential to know the amount of degeneration the patient has before treatment because sometimes excessive cone degeneration makes it impossible to fix blindness due to a CEP290 mutation. Something else that must be considered is the fact that the Lentivirus can cause insertional mutagenesis. Insertional mutagenesis is an issue because it causes abnormal growth. Although this is a problem, it is easily treated by laser photocoagulation without having a negative effect on the eye itself. Something else that geneticists have been working on is finding synthetic promoters that are highly specific because the human promoter has not been identified yet. Using a CMV promoter works, but there is hope that these synthetic promoters will allow for transgene expression of specific cells. The inserted CEP290 gene created cilia that were actually longer than the normal cilia from a normally functioning CEP290 gene. This does not have a significant impact on the ability for the cilia to function properly. As long as the amount of cells has increased to a number that allows for proper signal transduction, the gene therapy method is effective. References 1. Leber Congenital Amaurosis 2. [http://abstracts.iovs.org/cgi/content/short/54/6/2730 Gene Therapy for CEP290-associated LCA in Patient-Derived Induced Pluripotent Stem Cells] 3. Centrosomal-Ciliary Gene CEP290/NPHP6 Mutations Result in Blindness With Unexpected Sparing of Photoreceptors and Visual Brain: Implications for Therapy of Leber Congenital Amaurosis 4. Mutations in the CEP290 (NPHP6) Gene Are a Frequent Cause of Leber Congenital Amaurosis (PMCID: PMC1559533) 5. Unexpected CEP290 mRNA Splicing in a Humanized Knock-In Mouse Model for Leber Congenital Amaurosis(PMCID: PMC3819269) 6. CEP290gene transfer rescues Leber congenital amaurosis cellular phenotype